Transdifferentiation of Rat Keratinocyte Progenitors to Corneal Epithelial Cells by Limbal Niche via the STAT3/PI3K/AKT signaling pathway

PURPOSE. To develop a method for enriching keratinocyte progenitor cells (KPCs) and establish a limbal niche (LN)-mediated transdifferentiation protocol to induce KPCs transdifferentiation into corneal epithelial cells. METHODS. Limbal niche cells (LNCs) were isolated from limbal tissues through enzymatic digestion and characterized. Conditioned medium from LNCs cultures was collected. KPCs were enriched by rapid adhesion of Matrigel and subsequently cultured in either an LNCs-conditioned medium supplemented with KSFM (LN-KS) or SHEM (LN-SH) for 14 days. Corneal-specific marker expression was assessed to evaluate transdifferentiation efficiency. Key transcription factors and signaling pathways involved in the transdifferentiation process were identified through single-cell and RNA sequencing, and validated using western blot and quantitative real-time PCR. RESULTS. Both LN-KS and LN-SH protocols successfully induced corneal epithelial cell transdifferentiation from KPCs, with LN-KS demonstrating higher efficiency in generating CK12+ and p63+ cells (p<0.001). RNA sequencing analysis and western blot have revealed significant activation of STAT3 and PI3K/AKT signaling pathways. Inhibition of STAT3 blocked PI3K/AKT activation and impaired corneal epithelial cell transdifferentiation. CONCLUSIONS. This study demonstrates the ability of LN to promote KPCs transdifferentiation into corneal epithelial cells in vitro, a process partially mediated by the STAT3 and PI3K/AKT signaling pathways. Primary keratinocyte progenitors were transferred to Matrigel-coated six-well plates for 14 days, with daily medium changes. This induction phase was conducted under one of three culture conditions: (1) KSFM condition, employing commercial keratinocyte serum-free medium supplemented with rhEGFand BPE; (2) LNCs-KS condition, utilizing KSFM supplemented with LNCs conditioned medium at a 1:1 ratio; (3) LNC-SH condition, employing supplemented hormonal epithelial medium(SHEM) supplemented with LNCs conditioned medium at a 1:1 ratio.